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Fusion protein with transdermal capability and interleukin-10 activity as well as coding gene and application thereof

An interleukin and fusion protein technology, which is applied in the fusion protein and its encoding gene and application fields, can solve the problems of systemic immunosuppression side effects, side effects, poor treatment effect, etc., and achieves easy purification, low preparation cost and short production cycle. Effect

Inactive Publication Date: 2011-02-02
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At the same time, in phase II clinical observation, it was also found that the treatment effect is poor if the dosage of IL-10 is small; while increasing the dosage will produce obvious side effects
The main reason is that injection is currently the only route of administration of protein drugs, and because IL-10 is a strong natural immunosuppressant, systemic injection can lead to systemic immunosuppression and side effects

Method used

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  • Fusion protein with transdermal capability and interleukin-10 activity as well as coding gene and application thereof
  • Fusion protein with transdermal capability and interleukin-10 activity as well as coding gene and application thereof
  • Fusion protein with transdermal capability and interleukin-10 activity as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Acquisition of a gene encoding a fusion protein with transdermal ability and interleukin-10 activity

[0057] PCR method is used to amplify the coding gene of the fusion protein with transdermal ability and interleukin-10 activity of the present invention, and the specific process comprises the following steps:

[0058] 1. Amplification of the α-Factor signal peptide fragment of pPIC9K

[0059] 1) The 3' end is connected with the human IL-10 gene sequence and the amplification of the α-factor (α-Factor) signal peptide fragment coding sequence with the Kex2 single protease cleavage site

[0060] Using the pPIC9K plasmid (purchased from invitrogen) as a template, under the guidance of primer P3 (5'-ATGGATCCAAACGATGAGATTTC-3') and primer P4 (5'-TGCCCTGGCCTGGGCTTCTTTTCTCGAGAGATACCC-3'), PCR amplification with a single protease cleavage site for Kex2 The coding sequence of the α-factor signal peptide fragment, the PCR reaction conditions are: first 94°C for 2 min...

Embodiment 2

[0085] Example 2, Expression of fusion protein gene with transdermal ability and interleukin 10 activity in Pichia pastoris and purification of expression product

[0086] 1. Construction of Pichia pastoris expression vector with interleukin-10 active fusion protein gene

[0087] The specific method for constructing the Pichia pastoris expression vector of the fusion protein gene with interleukin-10 activity is: use restriction endonuclease BamHI (purchased from Promega, article number R6021) and EcoRI (purchased from Promega, article number R6011) The PCR products "α-Factor-IL10-Fc-γ1" and "α-Factor-TD1-IL10-Fc-γ1" obtained in Example 1 were subjected to double enzyme digestion, and then the digested fragments were respectively digested with the same enzyme double enzyme The excised plasmid pPIC9K was ligated with T4 DNA ligase (purchased from Shanghai Sangong, Cat. No. EL0015), and the ligated products were respectively transformed into E. coli DH5α competent cells, positive...

Embodiment 3

[0111] Example 3, IL-10 activity detection of fusion protein

[0112] Peripheral blood lymphocyte (PBMC) medium RPMI-1640 (purchased from HyClone, product number SH30809.01B);

[0113] Take one unit of peripheral blood from healthy people (purchased from Hefei Blood Bank), use Ficoll (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number: LTS1077) and separate peripheral blood lymphocytes (PBMC) according to its instructions. Add 0.1ml human PBMC (2×10 5 cells), the fusion protein IL10-IgG / Fc-γ1 purified by S200 molecular sieve chromatography obtained in Example 2, the fusion protein TD1-IL10-IgG / Fc-γ1 purified by S200 molecular sieve chromatography and commercial RhIL-10 (purchased from Rochy Hill Company, product number: 200-10), three duplicate wells were set up for each dilution, and after culturing at 37°C with 5% CO2 for 24h, the final concentration of 0.1ug / ml was added. PHA-P (purchased from sigma, product number: L8754), and blank c...

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Abstract

The invention discloses a fusion protein with transdermal capability and interleukin 10 activity as well as a coding gene and application thereof. The fusion protein provided by the invention is formed by connecting any one protein of (1) human IgG Fc-gamma1 or the mutain thereof and (2) human IgG Fc-gamma2 or the mutain thereof on the carboxyl terminal of a human interleukin 10 protein, and connecting a transit peptide on an amino terminal; wherein the amino acid sequence of the human interleukin 10 protein is the amino acid sequence from the 12th site to the 171st site on the amino terminal of a sequence 2 in a sequence table; and the amino acid sequence of the transit peptide is the amino acid sequence from the 1st site to the 11th site on the amino terminal of the sequence 2 in the sequence table. Tests prove that the fusion protein provided in the invention has the transdermal capability and the interleukin 10 activity simultaneously, and has greater practical significance and broad application prospect in the field of the preparation of medicaments for treating autoimmunity reaction and especially psoriasis.

Description

technical field [0001] The present invention relates to a fusion protein and its coding gene and application, in particular to a fusion protein with "transit peptide" and human interleukin-10 activity and its coding gene, as well as the expression method of the fusion protein and its preparation for the treatment of autoimmunity. Sexual response especially in the application of psoriasis drugs. Background technique [0002] Interleukin-10 (Interleukin-10, IL-10) is a multifunctional cytokine that can inhibit the immune inflammatory response mediated by T cells and participate in the regulation of humoral immunity. Studies have shown that IL-10 plays a key role in the initiation and development of tissue-specific autoimmune damage, and has been applied to diseases including type 1 diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, hepatitis C Clinical experimental research on various immune diseases. Among them, the treatment effect on psoriasis is the most obvi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61P37/02A61K38/20C12N5/10C12N1/19A61K47/48C07K1/22C07K1/16C12N15/62C07K19/00A61P17/06C12N1/15A61K48/00C12N15/63C12P21/02A61K47/42
Inventor 肖卫华郭雨刚温龙平张力康文瑶满娜李光伟
Owner UNIV OF SCI & TECH OF CHINA
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